Altered antibodies and their preparation

ABSTRACT

An altered antibody chain is produced in which the CDR&#39;s of the variable domain of the chain are derived from a first mammalian species. The framework-encoding regions of DNA encoding the variable domain of the first species are mutated so that the mutated framework-encoding regions encode a framework derived from a second different mammalian species. The or each constant domain of the antibody chain, if present, are also derived from the second mammalian species.

This application is a 371 of PCT/GB91/01578, filed Sep. 16, 1991.More than one reissue application has been filed for the reissue of U.S. Pat. No. 6,767,996. This application (Ser. No. 13/683,113) is a reissue application of U.S. Pat. No. 6,767,996 and is a continuation reissue of U.S. Reissue application Ser. No. 11/493,016 (now RE43,898), which is also a reissue of U.S. Pat. No. 6,767,996. U.S. Pat. No. 6,767,996 is issued from U.S. application Ser. No. 08/030,175 filed on May 17, 1993, which is the U.S. national stage application of PCT/GB91/01578, filed 16 Sep. 1991. PCT/GB91/01578 claims priority to UK Application No. 9020282.1 filed 17 Sep. 1990, all of which are incorporated herein in their entireties.

The present invention relates to altered antibodies and their preparation. The invention is typically applicable to the production of humanised antibodies.

Antibodies typically comprise two heavy chains linked together by disulphide bonds and two light chains. Each light chain is linked to a respective heavy chain by disulphide bonds. Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end. The light chain variable domain is aligned with the variable domain of the heavy chain. The light chain constant domain is aligned with the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.

The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.

The preparation of an altered antibody in which the CDRs are derived from a different species than the framework of the antibody's variable domains is disclosed in EP-A-0239400. The CDRs may be derived from a rat or mouse monoclonal antibody. The framework of the variable domains, and the constant domains, of the altered antibody may be derived from a human antibody. Such a humanised antibody elicits a negligible immune response when administered to a human compared to the immune response mounted by a human against a rat or mouse antibody. Humanised CAMPATH-1 antibody is disclosed in EP-A-0328404.

We have now devised a new way of preparing an altered antibody. In contrast to previous proposals, this involves altering the framework of a variable domain rather than the CDRs. This approach has the advantages that it does not require a pre-existing cDNA encoding, for example, a human framework to which to reshape and that it is technically easier than prior methodologies.

Accordingly, the present invention provides a process for the preparation of an antibody chain in which the CDRs of the variable domain of the antibody chain are derived from a first mammalian species and the framework of the variable domain and, if present, the or each constant domain of the antibody chain are derived from a second different mammalian species, which process comprises:

-   -   (i) mutating the framework-encoding regions of DNA encoding a         variable domain of an antibody chain of the said first species         such that the mutated framework-encoding regions encode the said         framework derived from the said second species; and     -   (ii) expressing the said antibody chain utilising the mutated         DNA from step (i).

A variable domain of either or both chains of an antibody can therefore be altered by:

-   -   (a) determining the nucleotide and predicted amino acid sequence         of a variable,domain of a selected antibody chain of the said         first species;     -   (b) determining the antibody framework to which the framework of         the said variable domain is to be altered;     -   (c) mutating the framework-encoding regions of DNA encoding the         said variable domain such that the mutated framework-encoding         regions encode the framework determined upon in step (b);     -   (d) linking the mutated DNA obtained in step (c) to DNA encoding         a constant domain of the said second species and cloning the DNA         into an expression vector; and     -   (e) introducing the expression vector into a compatible host         cell and culturing the host cell under such conditions that         antibody chain is expressed.

The antibody chain may be co-expressed with a complementary antibody chain. At least the framework of the variable domain and the or each constant domain of the complementary chain generally are derived from the said second species also. A light chain and a heavy chain may be co-expressed. Either or both chains may have been prepared by the process of the invention. Preferably the CDRs of both chains are derived from the same selected antibody. An antibody comprising both expressed chains can be recovered.

The antibody preferably has the structure of a natural antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fab′)₂ fragment, a Fab fragment, a light chain dimer or a heavy chain. The antibody may be an IgG such as an IgG1, IgG2, IgG3 or IgG4 IgM, IgA, IgE or IgD. Alternatively, the antibody may be a chimaeric antibody of the type described in WO 86/01533.

A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically, the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobulin region is fused at its C-terminus to the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence.

The invention is preferably employed to humanise an antibody, typically a monoclonal antibody and, for example, a rat or mouse antibody. The framework and constant domains of the resulting antibody are therefore human framework and constant domains whilst the CDRs of the light and/or heavy chain of the antibody are rat or mouse CDRs. Preferably all CDRs are rat or mouse CDRs. The antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat or mouse CDRs.

The process of the invention is carried out in such a way that the resulting antibody retains the antigen binding capability of the antibody from which it is derived. An antibody is reshaped according to the invention by mutating the framework-encoding regions of DNA coding for the variable domains of the antibody. This antibody and the reshaped antibody should both be capable of binding to the same antigen.

The starting antibody is typically an antibody of a selected specificity. In order to ensure that this specificity is retained, the variable domain framework of the antibody is preferably reshaped to about the closest variable domain framework of an antibody of another species. By “about the closest” is meant about the most homologous in terms of amino acid sequences. Preferably there is a homology of at least 50% between the two variable domains.

There are four general steps to reshape a monoclonal antibody. These are:

-   -   (1) determining the nucleotide and predicted amino acid sequence         of the starting antibody light and heavy chain variable domains;     -   (2) designing the reshaped antibody, i.e. deciding which         antibody framework region to use during the reshaping process;     -   (3) the actual reshaping methodologies/techniques; and     -   (4) the transfection and expression of the reshaped antibody.

These four steps are explained below in the context of humanising an antibody. However, they may equally well be applied when reshaping to an antibody of a non-human species.

Step 1: Determining the Nucleotide and Predicted Amino Acid Sequence of the Antibody Light and Heavy Chain Variable Domains

To reshape an antibody only the amino acid sequence of antibody's heavy and light chain variable domains needs to be known. The sequence of the constant domains is irrelevant because these do not contribute to the reshaping strategy. The simplest method of determining an antibody's variable domain amino acid sequence is from cloned cDNA encoding the heavy and light chain variable domain.

There are two general methods for cloning a given antibody's heavy and light chain variable domain cDNAs: (1) via a conventional cDNA library, or (2) via the polymerase chain reaction (PCR). Both of these methods are widely known. Given the nucleotide sequence of the cDNAs, it is a simple matter to translate this information into the predicted amino acid sequence of the antibody variable domains.

Step 2: Designing the Reshaped Antibody

There are several factors to consider in deciding which human antibody sequence to use during the reshaping. The reshaping of light and heavy chains are considered independently of one another, but the reasoning is basically similar for each.

This selection process is based on the following rationale: A given antibody's antigen specificity and affinity is primarily determined by the amino acid sequence of the variable region CDRs. Variable domain framework residues have little or no direct contribution. The primary function of the framework regions is to hold the CDRs in their proper spacial orientation to recognize antigen. Thus the substitution of rodent CDRs into a human variable domain framework is most likely to result in retention of their correct spacial orientation if the human variable domain is highly homologous to the rodent variable domain from which they originated. A human variable domain should preferably be chosen therefore that is highly homologous to the rodent variable domain(s).

A suitable human antibody variable domain sequence can be selected as follows:

-   1. Using a computer program, search all available protein (and DNA)     databases for those human antibody variable domain sequences that     are most homologous to the rodent antibody variable domains. This     can be easily accomplished with a program called FASTA but other     suitable programs are available. The output of a suitable program is     a list of sequences most homologous to the rodent antibody, the     percent homology to each sequence, and an alignment of each sequence     to the rodent sequence. This is done independently for both the     heavy and light chain variable domain sequences. The above analyses     are more easily accomplished if customized sub-databases are first     created that only include human immunoglobulin sequences. This has     two benefits. First, the actual computational time is greatly     reduced because analyses are restricted to only those sequences of     interest rather than all the sequences in the databases. The second     benefit is that, by restricting analyses to only human     immunoglobulin sequences, the output will not be cluttered by the     presence of rodent immunoglobulin sequences. There are far more     rodent immunoglobulin sequences in databases than there are human. -   2. List the human antibody variable domain sequences that have the     most overall homology to the rodent antibody variable domain (from     above). Do not make a distinction between homology within the     framework regions and CDRs. Consider the overall homology. -   3. Eliminate from consideration those human sequences that have CDRs     that are a different length than those of the rodent CDRs. This     rule-does not apply to CDR 3, because the length of this CDR is     normally quite variable. Also, there are sometimes no or very few     human sequences that have the same CDR lengths as that of the rodent     antibody. If this is the case, this rule can be loosened, and human     sequences with one or more differences in CDR length can be allowed. -   4. From the remaining human variable domains, the one is selected     that is most homologous to that of the rodent. -   5. The actual reshaped antibody (the end result) should contain CDRs     derived from the rodent antibody and a variable domain framework     from the human antibody chosen above.     Step 3: The Actual Reshaping Methodologies/techniques

A cDNA encoding the desired reshaped antibody is preferably made beginning with the rodent cDNA from which the rodent antibody variable domain sequence(s) was originally determined. The rodent variable domain amino acid sequence is compared to that of the chosen human antibody variable domain sequence. The residues in the rodent variable domain framework are marked that need to be changed to the corresponding residue in the human to make the rodent framework identical to that of the human framework. There may also be residues that need adding to or deleting from the rodent framework sequence to make it identical to that of the human.

Oligonucleotides are synthesised that can be used to mutagenize the rodent variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.

The advantages of this method of reshaping as opposed to splicing CDRs into a human framework are that (1) this method does not require a pre-existing cDNA encoding the human framework to which to reshape and (2) splicing CDRs is technically more difficult because there is usually a large region of poor homology between the mutagenic oligonucleotide and the human antibody variable domain. This is not so much a problem with the method of splicing human framework residues onto a rodent variable domain because there is no need for a pre-existing cDNA encoding the human variable domain. The method starts instead with the rodent cDNA sequence. Also, splicing framework regions is technically easier because there is a high degree of homology between the mutagenic oligonucleotide and the rodent variable domain framework. This is true because a human antibody variable domain framework has been selected that is most homologous to that of the rodent.

The advantage of the present method of reshaping as opposed to synthesizing the entire reshaped version from scratch is that it is technically easier. Synthesizing a reshaped variable domain from scratch requires several more oligonucleotides, several days more work, and technical difficulties are more likely to arise.

Step 4: The Transfection and Expression of the Reshaped Antibody

Following the mutagenesis reactions to reshape the antibody, the cDNAs are linked to the appropriate DNA encoding light or heavy chain constant region, cloned into an expression vector, and transfected into mammalian cells. These steps can be carried out in routine fashion. A reshaped antibody may therefore be prepared by a process comprising:

-   -   a) preparing a first replicable expression vector including a         suitable promoter operably linked to a DNA sequence which         encodes at least a variable domain of an Ig heavy or light         chain, the variable domain comprising framework regions from a         first antibody and CDRs comprising at least parts of the CDRs         from a second antibody of different specificity;     -   b) if necessary, preparing a second replicable expression vector         including a suitable promoter operably linked to a DNA sequence         which encodes at least the variable domain of a complementary Ig         light or heavy chain respectively;     -   c) transforming a cell line with the first or both prepared         vectors; and     -   d) culturing said transformed cell line to produce said altered         antibody.

Preferably the DNA sequence in step a) encodes both the variable domain and the or each constant domain of the antibody chain, the or each constant domain being derived from the first antibody. The antibody can be recovered and purified. The cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus. Most preferably, the immortalised cell line is a myeloma cell line or a derivative thereof.

Although the cell line used to produce the altered antibody is preferably a mammalian cell line, any other suitable cell line, such as a bacterial cell line or a yeast cell line, may alternatively be used. In particular, it is envisaged that E. coli—derived bacterial strains could be used.

It is known that some immortalised lymphoid cell lines, such as myeloma cell lines, in their normal state secrete isolated Ig light or heavy chains. If such a cell line is transformed with the vector prepared in step (a) it will not be necessary to carry out step (b) of the process, provided that the normally secreted chain is complementary to the variable domain of the Ig chain encoded by the vector prepared in step (a).

However, where the immortalised cell line does not secrete or does not secrete a complementary chain, it will be necessary to carry out step (b). This step may be carried out by further manipulating the vector produced in step (a) so that this vector encodes not only the variable domain of an altered antibody light or heavy chain, but also the complementary variable domain.

Alternatively, step (b) is carried out by preparing a second vector which is used to transform the immortalised cell line. This alternative leads to easier construct preparation, but may be less preferred than the first alternative in that it may not lead to as efficient production of antibody.

In the case where the immortalised cell line secretes a complementary light or heavy chain, the transformed cell line may be produced for example by transforming a suitable bacterial cell with the vector and then fusing the bacterial cell with the immortalised cell Line by spheroplast fusion. Alternatively, the DNA may be directly introduced into the immortalised cell line by electroporation or other suitable method.

An antibody is consequently produced in which CDRs of a variable domain of an antibody chain are homologous with the corresponding CDRs of an antibody of a first mammalian species and in which the framework of the variable domain and the constant domains of the antibody are homologous with the corresponding framework and constant domains of an antibody of a second, different, mammalian species. Typically, all three CDRs of the variable domain of a light or heavy chain are derived from the first species.

The present process has been applied to obtain an antibody against human CD4 antigen. Accordingly, the invention also provides an antibody which is capable of binding to human CD4 antigen, in which the CDRs of the light chain of the antibody have the amino acid sequences:

CDR1: LASEDIYSDLA (SEQ ID NO:13)

CDR2: NTDTLQN (SEQ ID NO:14)

CDR3: QQYNNYPWT (SEQ ID NO:15),

in which the CDRs of the heavy chain of the antibody have the amino acid sequences:

CDR1: NYGMA (SEQ ID NO:16)

CDR2: TISHDGSDTYFRDSVKG (SEQ ID NO:17)

CDR3: QGTIAGIRH (SEQ ID NO:18), and

in which the framework of the variable domain and, if present, the or each constant domain of each chain are derived from a mammalian non-rat species.

The antibody preferably has the structure of a natural antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fab′)₂ fragment, a Fab fragment, a light chain dimer or a heavy chain.

The antibody may be an IgG such as IgG1, IgG2, IgG3 or IgG4 IgM, IgA, IgE or IgD. Alternatively, the antibody may be a chimaeric antibody of the type described in WO 86/01533.

A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobulin region is fused at its C-terminus to the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence.

The invention is preferably employed to humanise a CD4 antibody such as a rat or mouse CD4 antibody. The framework and the constant domains of the resulting antibody are therefore human framework and constant domains whilst the CDRs of the light and/or heavy chain of the antibody are rat or mouse CDRs. Preferably all CDRs are rat or mouse CDRs. The antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or 1gD carrying rat or mouse CDRs.

Preferably the framework of the antibody heavy chain is homologous to the corresponding framework of the human antibody KOL (Schmidt et al, Hoppe-Seyler's Z. Physiol. Chem., 364 713-747, 1983). The sixth residue of framework 4 in this case is suitably Thr or Pro, preferably Thr. This residue is the 121st residue in the KOL antibody heavy chain variable region (Schmidt et al, 1983), and is identified as residue 108 by Kabat (Kabat et al, “Sequences of proteins of immunological interest”, US Dept of Health and Human Services, US Government Printing Office, 1987). Alternatively, the framework of the antibody heavy chain is homologous to the corresponding framework of the human antibody NEW (Saul et al, J. Biol. Chem. 2: 585-597, 1978). The final residue of framework 1 in this case is suitably Ser or Thr, preferably Ser. This residue is at position 30 (Kabat et al, 1987). Preferably the framework of the antibody light chain is homologous to the variable domain framework of the protein REI (Epp et al, Eur. J. Biochem., 45, 513-524, 1974).

The framework regions of one or both chains of a CD4 antibody can be reshaped by the present process. Alternatively, one or both chains of a CD4 antibody may be reshaped by the procedure described in EP-A-0239400. The procedure of EP-A-0239400 involves replacing CDRs rather than the replacement of frameworks. The CDRs are grafted onto a framework derived from a mammalian non-rat species, typically a human. This may be achieved by oligonucleotide-directed in vitro mutagenesis of the CDR-encoding regions of an antibody chain, light or heavy, from a mammalian non-rat species. The oligonucleotides in such an instance are selected so that the resulting CDR-grafted antibody has the light chain CDRs 1 to 3 and the heavy chain CDRs 1 to 3 shown above.

The reshaped CD4 antibody can be used to induce tolerance to an antigen. It can be used to alleviate autoimmune diseases such as rheumatoid arthritis. It can be used to prevent graft rejection. Tolerance to a graft such as an organ graft or a bone marrow transplantation can be achieved. Also, the reshaped CD4 antibody might be used to alleviate allergies. Tolerance to allergens could be achieved.

The CD4 antibody may be depleting or non-depleting. A depleting antibody is an antibody which depletes more than 50%, for example from 90 to 99%, of target cells in vivo. A non-depleting antibody depletes fewer than 50%, for example, from 10 to 25% and preferably less than 10% of target cells in vivo. A CD4 antibody may be administered alone or may be co-administered with a non-depleting or depleting CD8 antibody. The CD4 antibody, depleting or non-depleting, and CD8 monoclonal antibody, depleting or non-depleting, may be administered sequentially in any order or may be administered simultaneously. An additional antibody, drug or protein may be administered before, during or after administration of the antibodies.

A CD4 antibody and, indeed, a CD8 antibody as appropriate are given parenterally, for example intravenously. The antibody may be administered by injection or by infusion. For this purpose the antibody is formulated in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent. Any appropriate carrier or diluent may be employed, for example phosphate-buffered saline solution.

The amount of non-depleting or depleting CD4 and, if desired, CD8 antibody administered to a patient depends upon a variety of factors including the age and weight of a patient, the condition which is being treated and the antigen(s) to which it is desired to induce tolerance. In a model mouse system from 1 μg to 2 mg, preferably from 400 μg to 1 mg, of a mAb is administered at any one time. In humans from 3 to 500 mg, for example from 5 to 200 mg, of antibody may be administered at any one time. Many such doses may be given over a period of several weeks, typically 3 weeks.

A foreign antigen(s) to which it is desired to induce tolerance can be administered to a host before, during, or after a course of CD4 antibody (depleting or non-depleting) and/or CDs antibody (depleting or non-depleting). Typically, however, the antigen(s) is administered one week after commencement of antibody administration, and is terminated three weeks before the last antibody administration.

Tolerance can therefore be induced to an antigen in a host by administering non-depleting or depleting CD4 and CD8 mAbs and, under cover of the mAbs, the antigen. A patient may be operated on surgically under cover of the non-depleting or depleting CD4 and CD8 mAbs to be given a tissue transplant such as an organ graft or a bone marrow transplant. Also, tolerance may be induced to an antigen already possessed by a subject. Long term specific tolerance can be induced to a self antigen or antigens in order to treat autoimmune disease such as multiple sclerosis or rheumatoid arthritis. The condition of a patient suffering from autoimmune disease can therefore be alleviated.

The following Example illustrates the invention. In the accompanying drawings:

FIGS. 1-1A: shows the nucleotide and predicted amino acid sequence of rat CD4 antibody light chain variable region. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. Base pairs 1-269 (HindIII-PvuII) and 577-620 ([Bg1II/Bc1I]-BamHI) are part of the vector M13V_(K)PCR3, while base pairs 270-576 are from the PCR product of the CD4 antibody light chain variable region (V_(L)). CDRs (boxes) were identified by comparison to known immunological sequences (Kabat et al, “Sequences of proteins of immunological interest, US Dept of Health and Human Services, US Government Printing Office, 1987). The nucleotide sequence of FIG. 1 corresponds to SEQ ID NO:1.

FIGS. 2-2A: shows the nucleotide and predicted amino acid sequence of the reshaped CAMPATH-1 antibody light chain cDNA. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 2 corresponds to SEQ ID NO:2.

FIGS. 3-3A: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody light chain cDNA CD4V_(L)REI. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 3 corresponds to SEQ ID NO:3.

FIGS. 4-4A: shows the nucleotide and predicted amino acid sequence of rat CD4 antibody heavy chain variable region. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. Base pairs 1-272 (HindIII-Pstl) and 603-817 (BstEII-BamHI) are part of the vector M13V_(H)PCR1, while base pairs 273-602 are from the PCR product of the CD4 antibody heavy chain variable region (V_(H)). The nucleotide sequence of FIG. 4 corresponds to SEQ ID NO:4.

FIGS. 5, 5A-D: shows the nucleotide and predicted amino acid sequence of the reshaped CAMPATH-1 antibody heavy chain cDNA. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 5 corresponds to SEQ ID NO:5.

FIGS. 6, 6A-D: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain cDNA CD4V_(H)NEW-Thr³⁰. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 6 corresponds to SEQ ID NO:6.

FIGS. 7, 7A-D: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain cDNA CD4V_(H)NEW-Ser³⁰. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 7 corresponds to SEQ ID NO:7.

FIG. 8: shows the heavy chain variable (V) region amino acid sequence of the human myeloma protein KOL. CDRs are identified by boxes. This sequence is taken from the Swiss-Prot protein sequence database. The nucleotide sequence of FIG. 8 corresponds to SEQ ID NO:8.

FIGS. 9-9A: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V_(H)KOL-Pro¹¹³. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 9 corresponds to SEQ ID NO:9.

FIGS. 10-10A: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V_(H)KOL-Pro¹¹³ without immunoglobulin promoter. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 10 corresponds to SEQ ID NO:10.

FIGS. 11-11A: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V_(H)KOL-Thr¹¹³. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 11 corresponds to SEQ ID NO:11.

FIGS. 12-12A: shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V_(H)KOL-Thr¹¹³ without immunoglobulin promoter. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. The nucleotide sequence of FIG. 12 corresponds to SEQ ID NO:12.

FIG. 13: shows the results of an ELISA that compares the avidity of YNB46.1.8 and CD4V_(H)KOL-Thr¹¹³ antibodies. The X-axis indicates the concentration (μg/ml) of YNB46.1.8 (triangles) or CD4V_(H)KOL-Thr¹¹³ (circles) antibody. The Y-axis indicates the optical density at 492 nanometers.

EXAMPLE 1. Materials and Methods

Isolation of monoclonal antibody. The rat-derived anti-human CD4 antibody, clone YNB46.1.8 (IgG_(2b), kappa light chain serotype), was the result of fusion between a rat splenocyte and the Lou strain rat myeloma cell line Y3-Ag 1.2.3 (Galfre et al, Nature, 277: 131-133, 1979) and was selected by its binding to a rat T cell line NB2-6TG stably transfected with an expression vector containing a complementary DNA (cDNA) encoding the human CD4 antigen (Madden et al, Cell, 42: 93-104, 1985). Antibody was purified by high pressure liquid chromatography (HPLC).

Isolation of Antibody Variable Regions. cDNAs encoding the V_(L) and V_(H) regions of the CD4 antibody were isolated by a polymerase chain reaction (PCR)-based method (Orlandi et al, PNAS USA, 86: 3833-3837, 1989) with some modifications. Total RNA was isolated from hybridoma cells by the guanidine thiocyanate method (Chirgwin et al, Biochemistry, 18: 5294, 1979), and poly(A)⁺ RNA was isolated by passage of total RNA through and elution from an oligo(dT)-cellulose column (Aviv and Leder PNAS USA 69: 1408, 1972). Poly(A)⁺ RNA was heated at 70° C. for 5 minutes and cooled on ice just prior to use. A 25 μl first strand synthesis reaction consisted of 5 μg poly(A)⁺ RNA, 250 μM each dNTP, 50 mM Tris.HCl (pH 8.2 at 42° C.), 10 mM MgCl₂, 100 mM KCl, 10 mM dithiothreitol, 23 units reverse transcriptase (Anglian Biotec, Colchester, U.K.), 3.5 pmoles of the V_(L) region-specific oligonucleotide primer V_(K)1FOR [5′-d(GTT AGA TCT CCA GCT TGG TCC C)SEQ ID NO:19] or the V_(H) region-specific prime V_(H)1FOR-B [5,-d(TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC)SEQ ID NO:20], and incubated for 5 minutes at 20° C. and then 90 minutes at 42° C.

Subsequent 50 μl PCR amplifications consisted of 5 μl of the first strand synthesis reaction (unpurified), 500 μM each dNTP, 67 mM Tris-HCl (pH 8.8 at 25° C.), 17 mM (NH₄)₂SO₄, 10 mM MgCl₂, 20 μg/ml gelatin, 5 units TAQ DNA polymerase (Koch-Light, Haverhill, U.K.), and 25 pmoles (each) of primer V_(K)1FOR and V_(K)1BACK [5′-d(GAC ATT CAG CTG ACC CAG TCT)SEQ ID NO:21] for the V_(L) region or V_(H)1FOR-B and the mixed primer V_(H)1BACK [5′-d(AG GT(CG) (CA)A(GA) CTG CAG (GC)AG TC(TA)GG)SEQ ID NO:22] for the V_(H) region. Reactions were overlayed with mineral oil and subjected to 30 cycles of 1.5 minutes at 95° C. (denaturation), 1.5 minutes at 37° C. (V_(L)) or 50° C. (V_(H); annealing), and 3 minutes at 72° C. (extension) with a Techne PHC-1 programmable cyclic reactor. The final cycle contained a 10 minute extension time.

The samples were frozen at −20° C. and the mineral oil (a viscous liquid at −20° C.) was removed by aspiration. The aqueous phases were thawed, and PCR products were purified by electrophoresis in 2% agarose gels, and then double digested with either PvuII and BglII (V_(L)) or PstI and BstEII (V_(H)) restriction enzymes, and cloned into the PvuII and BclI restriction sites of the vector M13V_(K)PCR3 (for V_(L) region; Orlandi et al, 1989) or the PstI and BstEII restriction sites of the vector M13V_(H)PCR1 (for V_(H) region). As described in the results, V_(L) region clones were first screened by hybridisation to a ³²P-labeled oligonucleotide probe [5′-d(GTT TCA TAA TAT TGG AGA CA)SEQ ID NO:23] for the CDR2 of the Y3-Ag 1.2.3 V_(L) region. V_(L) region clones not hybridising to this probe and V_(H) region clones were sequenced by the dideoxy chain termination method (Sanger et al, PNAS USA 74: 5463, 1977).

Reshaped Light Chain Variable Region and Expression Vector Construct

The reshaped light chain was constructed by oligonucleotide-directed in vitro mutagenesis in an M13 vector by priming with three oligonucleotides simultaneously on a 748 base single-stranded cDNA template encoding the entire V_(L) and kappa constant (C_(K)) regions of the reshaped CAMPATH-1 antibody (Reichmann et al, Nature 332: 323-327, 1988). The three oligonucleotides [5′-d(AGA GTG ACC ATC ACC TGT CTA GCA AGT GAG GAC ATT TAC AGT GAT TTA GCA TGG TAC CAG CAG AAG CCA)SEQ ID NO:24, 5′-d(CTG CTG ATC TAC AAT ACA GAT ACC TTG CAA AAT GGT GTG CCA AGC AGA TTC)SEQ ID NO:25, 5′-d(ATC GCC ACC TAC TAC TGC CAA CAG TAT AAC AAT TAT CCG TGG ACG TTC GGC CAA GGG ACC)SEQ ID NO:26] were designed to replace each of the three CDRs in the REI-based human antibody V_(L) region framework that is part of the reshaped CAMPATH-1 antibody V_(L) region (Reichmann et al, 1988). A clone containing each of the three mutant oligonucleotides was identified by nucleotide sequencing and was subcloned into the HindIII site of the expression vector pHβAPr-1 (Gunning et al, PNAS, 4: 4831-4835, 1987) which also contained a dihydrofolate reductase gene (Ringold et al, J. Mol. Appl. Genet. 1: 165-175, 1981) driven by a truncated SV40 promoter.

Reshaped Heavy Chain Variable Regions Based on the Variable Region Framework of the Human Antibody NEW, and Expression Vector Constructs

Two versions of the NEW-based reshaped heavy chain were created, CD4V_(H)NEW-Thr³⁰ and CD4V_(H)NEW-Ser³⁰. The CD4V_(H)NEW-Thr³⁰ version (FIG. 6) encodes a threonine residue at position 30 while the CD4V_(H)NEW-Ser³⁰ version (FIG. 7) encodes a Ser residue at position 30. As a matter of convenience, CD4V_(H)NEW-Thr³⁰ was created first by oligonucleotide-directed in vitro mutagenesis in the vector M13mp18 by priming with three oligonucleotides simultaneously on a 1467 base single-stranded cDNA template (FIG. 5) encoding the entire heavy chain of the reshaped CAMPATH-1 antibody (Reichmann et al, 1988). The three oligonucleotides [5′-d(TCT GGC TTC ACC TTC ACC AAC TAT GGC ATG GCC TGG GTG AGA CAG CCA CCT) SEQ ID NO:27, 5′-d(GGT CTT GAG TGG ATT GGA ACC ATT AGT CAT GAT GGT AGT GAC ACT TAC TTT CGA GAC TCT GTG AAG GGG AGA GTG)SEQ ID NO:28, 5′-d(GTC TAT TAT TGT GCA AGA CAA GGC ACT ATA GCT GGT ATA CGT CAC TGG GGT CAA GGC AGC CTC)SEQ ID NO:29] were designed to replace each of the three complementarity determining regions (CDRs) in the NEW-based V_(H) region that is part of the reshaped CAMPATH-1 antibody (Reichmann et al, 1988). A clone (FIG. 6) containing each of the three mutant oligonucleotides was identified by nucleotide sequencing. CD4V_(H)NEW-Ser³⁰ was created second by oligonucleotide-directed in vitro mutagenesis in the vector M13mp18 by priming with a single oligonucleotide on the 1458 base single-stranded cDNA template (FIG. 6) encoding CD4V_(H)NEW-Thr³⁰. Th oligonucleotide [5′-d(GCT TCA CCT TCA GCA ACT ATG GCA T)SEQ ID NO:30] was designed to mutate the residue at position 30 from threonine [ACC] to serine [AGC]. A clone (FIG. 7) containing this mutant oligonucleotide was identified by nucleotide sequencing. Double-stranded forms of the clones CD4V_(H)NEW-Thr³⁰ and CD4V_(H)NEW-Ser³⁰ were subcloned as HindIII fragments into the HindIII site of the expression vector pNH316. The vector pNH316 is a modified version of the vector pHβAPr-1 (Gunning et al, PNAS, 84: 4831-4835, 1987) which was engineered to contain a neomycin resistance gene driven by a metallothionine promoter.

Reshaped Heavy Chain Variable Regions Based on the Variable Region Framework of the Human Antibody KOL, and Expression Vector Constructs

Two versions of the KOL-based reshaped heavy chain were created, CD4V_(H)KOL-Thr¹¹³ and CD4V_(H)KOL-Pro¹¹³. The CD4V_(H)KOL-Thr¹¹³ version encodes a threonine residue at position 113 (FIG. 11) while the CD4V_(H)KOL-Pro¹¹³ version encodes a proline residue at position 113 (FIG. 9). As a matter of convenience, CD4V_(H)KOL-Thr¹¹³ was created first by oligonucleotide-directed in vitro mutagenesis of single-stranded DNA template containing the 817 base HindIII-BamHI fragment encoding the V_(H) region of the rat CD4 antibody (FIG. 4) cloned into M13mp18 by priming simultaneously with five oligonucleotides [5′-d(CAC TCC CAG GTC CAA CTG GTG GAG TCT GGT GGA GGC GTG GTG GAG CCT GG)SEQ ID NO:31, 5′-d(AAG GTC CCT GAG ACT CTC CTG TTC CTC CTC TGG ATT CAT CTT CAG TAA CTA TGG CAT G)SEQ ID NO:32, 5′-d(GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG)SEQ ID NO:33, 5′-d(ACT ATC TCC AGA GAT AAT AGC AAA AAC ACC CTA TTC CTG CAA ATG G)SEQ ID NO:34, 5′-d(ACA GTC TGA GGC CCG AGG ACA CGG GCG TGT ATT TCT GTG CAA GAC AAG GGA C)SEQ ID NO:35] which were designed to replace the rat framework regions with the human framework regions of KOL. A clone containing each of the five mutant oligonucleotides was identified by nucleotide sequencing. CD4V_(H)KOL-Pro¹¹³ was created second by oligonucleotide-directed in vitro mutagenesis of single-stranded DNA template containing the 817 base HindIII-BamHI fragment encoding CD4V_(H)KOL-Thr¹¹³ cloned into M13mp18 by priming with the oligonucleotide [5′-d(TGG GGC CAA GGG ACC CCC GTC ACC GTC TCC TCA)SEQ ID NO:36]. A clone containing this mutant oligonucleotide was identified by nucleotide sequencing.

The immunoglobulin promoters were removed from the double-stranded DNA forms of clones encoding CD4V_(H)KOL-Thr¹¹³ (FIG. 11) and CD4V_(H)KOL-Pro¹¹³ (FIG. 9) by replacing (for both versions) the first 125 bp (HindIII-NcoI) with a HindIII-NcoI oligonucleotide linker fragment [5′-d(AGC TTT ACA GTT ACT GAG CAC ACA GGA CCT CAC)SEQ ID NO:37 and its overlapping complement 5′-d(CAT GGT GAG GTC CTG TGT GCT CAG TAA CTG TAA)SEQ ID NO:38]. The resultant clones, CD4V_(H)KOL-Thr¹¹³ (FIG. 12) and CD4V_(H)KOL-Pro¹¹³ (FIG. 10), now 731 bp HindIII-BamHI fragments, were separately subcloned into the Hindlll and BamHI cloning sites of the expression vector pHβAPr-1-gpt (Gunning et al, PNAS USA 76, 1373, 1987) into which had been cloned the human IgG1 constant region gene (Bruggemann et al, J. Exp. Med. 166, 1351-1361, 1987) at the BamHI site. Thus, when transfected and expressed as antibody heavy chains (see below), these reshaped V_(H) regions are linked to human IgG1 constant regions.

Fluorescence Activated Cell Sorter (FACS) Analysis

The relative affinities of the reshaped antibodies to bind the CD4 antigen were estimated by FACS analysis. The CD4-expressing cells used in this analysis were a cloned rat T cell line NB2-6TG stabily transfected with an expression vector containing a complementary DNA (cDNA) encoding the human CD4 antigen (Maddon et al, Cell, 42, 93-104, 1985). Cells were stained with the appropriate reshaped antibody followed by fluorescein-conjugated sheep anti-human antibodies (Binding Site Ltd., Birmingham, UK). Control staining (see Table 1) consisted of no antibody present during the first stage of cell staining. Mean cellular fluorescence was determined with an Ortho FACS.

Antibody Avidity Analysis

The relative avidities of the rat YNB46.1.8 antibody and the reshaped CD4V_(H)KOL-Thr¹¹³ antibody were estimated by an enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated with soluble recombinant CD4 antigen (Byrn et al, Nature, 344: 667-670, 1990) at 50 ul/well, 10 ug/ml, and then blocked with 100 ul/well phosphate buffered saline (PBS) containing 1.0% bovine serum albumin (BSA). Antibodies were diluted in PBS containing 0.1% BSA, and added to wells (50 ul/well) for 45 minutes at room temperature. Biotinylated CD4V_(H)KOL-Thr113 antibody (10 ul/well; 20 ug/ml final concentration) was then added to each well for an additional 45 minutes. Wells were washed with PBS containing 0.1% BSA, and then 50 ul streptavidin-biotinylated horseradish peroxidase complex (Amersham; Aylesbury, UK) diluted 1:1,000 was added to each well for 30 minutes. Wells were washed with PBS containing 0.1% BSA, and 100 ul substrate (25 mM citric acid, 50 mM disodium hydrogen phosphate, 0.1% (w/v) o-phenylene diamine, 0.04% (v/v) 30% hydrogen peroxide) was added to each well. Reactions were stopped by the addition of 50 ul/well 1.0 M sulfuric acid. Optical densities at 492 nanometers (OD₄₉₂) were determined with an ELISA plate reader.

Transfections

Dihydrofolate reductase deficient chinese hamster ovary (CHO^(DHFR−)) cells (10⁶/T-75 flask) were cotransfected as described (Wigler et al, PNAS USA 76, 1373, 1979) with 9 μg of heavy chain construct and 1 μg of the light chain construct. Transfectants were selected in medium containing 5% dialysed foetal bovine serum for 2 to 3 weeks, and antibody-secreting clones were identified by ELISAs of conditioned media. Antibody was concentrated and purified by protein-A Sepharose (Trade Mark) column chromatography.

2. Results Cloning of Light and Heavy Chain Variable Region cDNAs

cDNAs encoding the V_(L) and V_(H) regions from CD4 antibody-secreting hybridoma cells were isolated by PCR using primers which amplify the segment of mRNA encoding the N-terminal region through to the J region (Orlandi et al, 1989). V_(L) and V_(H) region PCR products were subcloned into the M13-based vectors M13V_(K)PCR3 and M13V_(H)PCR1, respectively. Initial nucleotide sequence analysis of random V_(L) region clones revealed that most of the cDNAs encoded the V_(L) region of the light chain expressed by the Y3-Ag 1.2.3 rat myeloma cell line (Crowe et al, Nucleic Acid Research, 17: 7992, 1989) that was used as the fusion partner to generate the anti-CD4 hybridoma. It is likely that the expression of the Y3-Ag 1.2.3 light chain mRNA is greater than that of the CD4 antibody light chain, or the Y3-Ag 1.2.3 light chain mRNA is preferentially amplified during the PCR.

To maximize the chance of finding CD4 V_(L) region cDNAs, we first screened all M13 clones by hybridisation to a ³²P-labeled oligonucleotide probe that is complementary to the CDR 2 of Y3-Ag 1.2.3 (Crowe et al, Nucleic Acid Research, 17: 7992, 1989). Subsequent sequence analysis was restricted to M13 clones which did not contain sequence complementary to this probe. In this manner, two cDNA clones from independent PCR amplifications were identified that encoded identical V_(L) regions. Nucleotide sequence analysis of random V_(H) region PCR products revealed a single species of V_(H) region cDNA. Two V_(H) cDNA clones from independent PCR amplifications were found to contain identical sequences except that the codon of residue 14 encoded proline [CCT] in one clone while the second clone encoded leucine [CTT] at the same position.

According to Kabat et al 1987, 524 of 595 sequenced V_(H) regions contain a proline residue at this position, while only 6 contain leucine. We have therefore chosen the proline-encoding clone for illustration (see below). As residue 14 lies well within the first V_(H) framework region and not in a CDR, it is unlikely to contribute directly to antigen binding, and the ambiguity at this position did not affect the subsequent reshaping strategy. Thus, we have not investigated this sequence ambiguity further.

The cDNA sequences and their predicted amino acid sequences are shown in FIGS. 1 and 4. As no additional V_(L) or V_(H) region-encoding clones were found, it was assumed that these sequences were derived from the CD4 antibody genes.

Construction of Reshaped Antibodies

Our goal was to investigate the importance of selecting the appropriate human V region framework during reshaping. Two reshaping strategies were employed.

First Reshaping Strategy

In the first strategy, we created a reshaped antibody that incorporated the CDRs from the rat-derived CD4 antibody and the same human V region framework sequences that we had previously successfully used for the reshaped CAMPATH-1 antibody, namely an REI-based framework for the V_(L) region and an NEW-based framework for the V_(H) region (Reichmann et al, 1988). This was accomplished by oligonucleotide-directed in vitro mutagenesis of the six CDRs of the reshaped CAMPATH-1 antibody light and heavy chain cDNAs shown in FIGS. 2 and 5, respectively. The resultant reshaped CD antibody light chain (FIG. 3) is called CD4V_(L)REI. Two versions of the NEW-based reshaped CD4 antibody heavy chain were created: CD4V_(H)NEW-Thr³⁰ (FIG. 6) encoding a threonine residue at position 30 (in framework 1) and CD4V_(H)NEW-Ser³⁰ (FIG. 7) encoding a serine residue at position 30. These two different versions were created because the successfully reshaped CAMPATH-1 antibody heavy chain bound antigen well whether position 30 encoded a threonine or serine residue (Reichmann et al, 1988), and we chose to test both possibilities in this case as well.

Second Reshaping Strategy

In the second reshaping strategy, we have reshaped the CD4 antibody V_(H) region to contain the V_(H) region framework sequences of the human antibody KOL. Of all known human antibody V_(H) regions, the overall amino acid sequence of the V_(H) region of KOL is most homologous to the rat CD4 antibody V_(H) region. The V_(H) regions of the human antibodies KOL and NEW are 66% and 42% homologous to the rat CD4 antibody V_(H) region, respectively.

Two versions of the KOL-based reshaped CD4 antibody heavy chain V region were created that differ by a single amino acid residue within the fourth framework region: CD4V_(H)KOL-Pro¹¹³ (FIG. 10) encodes a proline residue at position 113 and CD4V_(H)KOL-Thr¹¹³ (FIG. 12) encodes a threonine residue at position 113. CD4V_(H)KOL-Pro¹¹³ is “true to form” in that its framework sequences are identical to those of the KOL antibody heavy chain V region (FIG. 8).

Of all known human antibody V_(L) regions, the overall amino acid sequence of the V_(L) region of the human light chain NEW is most homologous (67%) to the rat CD4 antibody V_(L) region. Thus, the identical reshaped light chain, CD4V_(L)REI (described above), that was expressed with the NEW-based reshaped CD4 antibody heavy chains CD4V_(H)NEW-Thr³⁰ and CD4V_(H)NEW-Ser³⁰, is also expressed with the KOL-based reshaped CD4 antibody heavy chains CD4V_(H)KOL-Pro¹¹³ and CD4V_(H)KOL-Thr¹¹³. This is advantageous because expression of the same reshaped light chain with different reshaped heavy chains allows for a direct functional comparison of each reshaped heavy chain.

To summarise, four different reshaped antibodies were created. The reshaped light chain of each antibody is called CD4V_(L)REI. The reshaped heavy chains of the antibodies are called CD4V_(H)NEW-Thr³⁰, CD4V_(H)NEW-Ser³⁰, CD4V_(H)KOL-Pro¹¹³, and CD4V_(H)KOL-Thr¹¹³, respectively. Each of the reshaped heavy chains contain the same human IgG1 constant region. As each reshaped antibody contains the same reshaped light chain, the name of a reshaped antibody's heavy chain shall be used below to refer to the whole antibody (heavy and light chain combination).

Relative Affinities of the Reshaped Antibodies

The relative affinities of the reshaped antibodies were approximated by measuring their ability to bind to CD4 antigen-expressing cells at various antibody concentrations. FACS analysis determined the mean cellular fluorescence of the stained cells (Table 1).

It is clear from this analysis that the reshaped CD4 antibodies bind to CD4 antigen to varying degrees over a broad concentration range. Consider Experiment 1 of Table 1 first. Comparing CD4V_(H)KOL-Thr¹¹³ antibody to CD4V_(H)NEW-Thr³⁰ antibody, it is clear that both antibodies bind CD4⁺ cells when compared to the control, reshaped CAMPATH-1 antibody. However, CD4V_(H)KOL-Thr¹¹³ antibody binds CD4⁺ cells with far greater affinity than CD4V_(H)NEW-Thr³⁰ antibody. The lowest concentration of CD4V_(H)KOL-Thr¹¹³ antibody tested (2.5 ug/ml) gave a mean cellular fluorescence nearly equivalent to that of the highest concentration of CD4V_(H)NEW-Thr³⁰ antibody tested (168 ug/ml). Experiment 2 demonstrates that CD4V_(H)NEW-Ser³⁰ antibody may bind CD4⁺ cells somewhat better than CD4V_(H)NEW-Thr³⁰. Only 2.5 ug/ml CD4V_(H)NEW-Ser³⁰ antibody is required to give a mean cellular fluorescence nearly equivalent to 10 ug/ml CD4V_(H)NEW-Thr³⁰ antibody. Experiment 3 demonstrates that CD4V_(H)KOL-Thr¹¹³ antibody may bind CD4⁺ cells somewhat better than CD4V_(H)KOL-Pro¹¹³ antibody.

From these assays, it is clear that the KOL-based reshaped antibodies are far superior to the NEW-based reshaped antibodies with regards to affinity towards CD4⁺ cells. Also, there is a lesser difference, if any, between CD4V_(H)NEW-Thr³⁰ antibody and CD4V_(H)NEW-Ser¹¹³ antibody, and likewise between CD4V_(H)KOL-Thr¹¹³ antibody and CD4V_(H)KOL-Pro¹¹³ antibody. A ranking of these reshaped antibodies can thus be derived based on their relative affinities for CD4+ cells:

-   -   CD4V_(H)KOL-Thr¹¹³→CD4V_(H)KOL-Pro¹¹³→CD4V_(H)NEW-Ser³⁰→CD4V_(H)NEW-Thr³⁰

It should be restated that each of the reshaped CD4 antibodies used in the above experiments have the identical heavy chain constant regions, and are associated with identical reshaped light chains. Thus observed differences of binding to CD4+ cells must be due to differences in their heavy chain V regions.

-   -   Relative Avidities of the Rat YNB46.1.8 Antibody and the         Reshaped CD4V_(H)KOL-Thr¹¹³ Antibody

The relative avidities of the rat YNB46.1.8 antibody and the reshaped CD4V_(H)KOL-Thr¹¹³ antibody were estimated by ELISA. In this assay, the ability of each antibody to inhibit the binding of biotinylated CD4V_(H)KOL-Thr¹¹³ antibody to soluble recombinant CD4 antigen was determined. Results of an experiment are shown in FIG. 13. The inhibition of binding of biotinylated CD4V_(H)KOL-Thr¹¹³ antibody was linear for both the unlabeled CD4V_(H)KOL-Thr¹¹³ and YNB46.1.8 antibodies near the optical density of 0.3. The concentrations of CD4V_(H)KOL-Thr¹¹³ and YNB46.1.8 antibodies that give an optical density of 0.3 are 28.7 and 1.56 ug/ml, respectively. Thus the avidity of the YNB46.1.8 antibody can be estimated to be 28.7/1.56 or about 18 times better than that of CD4V_(H)KOL-Thr¹¹³ antibody. It should be noted that this experiment only provides a rough approximation of relative avidities, not affinities. The rat YNB46.1.8 antibody contains a different constant region than that of the CD4V_(H)KOL-Thr¹¹³ antibody, and this could affect how well the antibodies bind CD4 antigen, irrespective of their actual affinities for CD4 antigen. The actual affinity of the reshaped antibodies for CD4 antigen may be greater, lesser, or the same as the YNB46.1.8 antibody. The other reshaped antibodies CD4V_(H)KOL-Pro¹¹³, CD4V_(H)NEW-Ser³⁰, and CD4V_(H)NEW-Thr³⁰ have not yet been tested in this assay.

TABLE 1 Mean cellular fluorescence of CD4⁺ cells stained with reshaped antibodies Concentration Mean cellular Reshaped Antibody (μg/ml) Fluorescence Experiment 1. CD4V_(H)KOL-Thr¹¹³ 113 578.0 CD4V_(H)KOL-Thr¹¹³ 40 549.0 CD4V_(H)KOL-Thr¹¹³ 10 301.9 CD4V_(H)KOL-Thr¹¹³ 2.5 100.5 CD4V_(H)NEW-Thr³⁰ 168 97.0 CD4V_(H)NEW-Thr³⁰ 40 40.4 CD4V_(H)NEW-Thr³⁰ 10 18.7 CD4V_(H)NEW-Thr³⁰ 2.5 10.9 CAMPATH-1 100 11.6 CAMPATH-1 40 9.4 CAMPATH-1 10 9.0 CAMPATH-1 2.5 8.6 CONTROL — 9.0 Experiment 2. CD4V_(H)NEW-Thr³⁰ 168 151.3 CD4V_(H)NEW-Thr³⁰ 40 81.5 CD4V_(H)NEW-Thr³⁰ 10 51.0 CD4V_(H)NEW-Thr³⁰ 2.5 39.3 CD4V_(H)NEW-Ser³⁰ 160 260.2 CD4V_(H)NEW-Ser³⁰ 40 123.5 CD4V_(H)NEW-Ser³⁰ 10 68.6 CD4V_(H)NEW-Ser³⁰ 2.5 49.2 CONTROL — 35.8 Experiment 3. CD4V_(H)KOL-Pro¹¹³ 100 594.9 CD4V_(H)KOL-Pro¹¹³ 40 372.0 CD4V_(H)KOL-Pro¹¹³ 10 137.7 CD4V_(H)KOL-Pro¹¹³ 2.5 48.9 CD4V_(H)KOL-Thr¹¹³ 100 696.7 CD4V_(H)KOL-Thr¹¹³ 40 631.5 CD4V_(H)KOL-Thr¹¹³ 10 304.1 CD4V_(H)KOL-Thr¹¹³ 2.5 104.0 CONTROL — 12.3 

What is claimed is:
 1. An antibody which is capable of binding to human CD4 antigen, in which the CDRs of the light chain of the antibody have the amino acid sequences: CDR1: LASEDIYSDLA (SEQ ID NO:13) CDR2: NTDTLQN (SEQ ID NO:14) CDR3: QQYNNYPWT (SEQ ID NO:15) in which the CDRs of the heavy chain of the antibody have the amino acid sequences: CDR1: NYGMA (SEQ ID NO:16) CDR2: TISHDGSDTYFRDSVKG (SEQ ID NO:17) CDR3: QGTIAGIRH (SEQ ID NO:18), and in which the framework of the variable domain of each chain and any constant domain present in said chain are derived from a mammalian non-rat species.
 2. An antibody according to claim 1, in which the mammalian non-rat species is human.
 3. An antibody according to claim 2, in which the variable domain framework region of the heavy chain consists essentially of the heavy chain variable domain framework region of the protein KOL.
 4. An antibody according to claim 3, in which the heavy chain variable domain has the amino acid sequence shown in the upper line in FIG. 10 (SEQ ID NO:39) or 12 (SEQ ID NO:40).
 5. An antibody according to claim 2, in which the variable domain framework region of the heavy chain consists essentially of the heavy chain variable domain framework region of the protein NEW.
 6. An antibody according to claim 5, in which the heavy chain variable domain has the amino acid sequence shown in the upper line of FIG. 6 (SEQ ID NO:41) or 7 (SEQ ID NO:42).
 7. An antibody according to claims 2, 3, 4, 5 or 6, in which the variable domain framework of the light chain consists essentially of the variable domain framework of the protein REI.
 8. An antibody according to claim 7, in which the light chain has the amino acid sequence shown in the upper line of FIG. 3 (SEQ ID NO:43).
 9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody as claimed in claim
 1. 10. An antibody which is capable of binding to human CD4 antigen, in which the CDRs of the light chain of the antibody have the amino acid sequences: CDR1: LASEDIYSDLA (SEQ ID NO:13) CDR2: NTDTLQN (SEQ ID NO:14) CDR3: QQYNNYPWT (SEQ ID NO:15), and in which the CDRs of the heavy chain of the antibody have the amino acid sequences: CDR1: NYGMA (SEQ ID NO:16) CDR2: TISHDGSDTYFRDSVKG (SEQ ID NO:17) CDR3: QCTIAGIRH (SEQ ID NO:18), and in which the framework of the variable domain of each chain and the constant region of said chain are derived from a human.
 11. An antibody according to claim 1, wherein the antibody has glycosylation characteristic of CHO cells.
 12. A humanized antibody, or an antigen-binding fragment of said humanized antibody, wherein the humanized antibody comprises (1) a light chain variable domain comprising a framework and three complementarity determining regions (CDRs) followed by constant regions and (2) a heavy chain variable domain comprising a framework and three CDRs followed by constant regions, wherein: the amino acid sequence of the CDRs consists of the amino acid sequence of the CDRs of an antibody of a first species, wherein the first species is a non-human mammal; the amino acid sequence of the light chain framework, of the heavy chain framework, or of both the light and the heavy chain frameworks consists of the amino acid sequence of a selected human antibody variable region, wherein the light chain framework consists of four framework regions and the heavy chain framework consists of four framework regions; wherein the humanized antibody differs from the antibody of a first species at least in that, for the humanized antibody: i.) the sequence of all four framework regions of the light chain is identical to the sequence of all four framework regions of a single selected human antibody variable region; or ii.) the sequence of all four framework regions of the heavy chain is identical to the sequence of all four framework regions of a single selected human antibody variable region; or iii.) both i.) and ii); and wherein: iv.) the selected human antibody variable region of i.) is selected from the human light chain antibody variable regions with the most overall homology to the light chain variable region of the antibody of the first species; or v.) the selected human antibody variable region of ii.) is selected from the human heavy chain antibody variable regions with the most overall homology to the heavy chain variable region of the antibody of the first species; or vi.) both iv.) and v.); and vii.); wherein the most overall homology is determined on the basis of the respective variable domains, considering the overall homology, without making a distinction between homology within the framework regions and CDRs, and wherein only CDR3 of either the selected human heavy chain and/or selected human light chain variable region has a different length as that of the corresponding CDRs of the first species antibody; wherein the humanized antibody is capable of binding the same human antigen as the antibody of the first species, wherein said antigen is not a CD antigen; wherein at least one of the constant regions is from a human immunoglobulin different from the antibody or antibodies from which the human frameworks are selected; and wherein the antibody of the first species is not an antibody that binds a CD antigen.
 13. A humanized antibody according to claim 12, or an antigen-binding fragment of said humanized antibody, wherein the non-human mammal is rat.
 14. A humanized antibody according to claim 12, or an antigen-binding fragment of said humanized antibody, wherein the non-human mammal is mouse.
 15. A humanized antibody according to claim 12, or an antigen-binding fragment of said humanized antibody, wherein the humanized antibody or fragment thereof is a (Fab′)₂ fragment.
 16. A humanized antibody according to claim 12, or an antigen-binding fragment of said humanized antibody, wherein the humanized antibody or fragment thereof is a Fab fragment.
 17. A humanized antibody according to claim 12, or an antigen-binding fragment of said humanized antibody, wherein the antibody has glycosylation characteristic of CHO cells.
 18. A pharmaceutical composition comprising a humanized antibody according to claim 12, or an antigen-binding fragment of said humanized antibody, and a pharmaceutically acceptable carrier or diluent. 